Organ fibrosis, a major pathological manifestation of scleroderma (SSc), is the result of excessive deposition of collagen I and other extracellular matrix (ECM) proteins. Despite the significant progress that has been made towards unraveling the physiological and pathological mechanisms involved in regulation of collagen genes, a full understanding of these processes is still lacking. In particular, very little is currently known about the molecular mechanism responsible for constitutive upregulation of ECM proteins by SSc fibroblasts. Such knowledge is critical for development of suitable targets for therapeutic intervention. During the last funding period we proposed to test the hypothesis that autocrine TGF-B signaling through overexpression of TGFB receptors is at least partially responsible for the SSc phenotype. To test this hypothesis we blocked TGF-B signaling by overexpressing a kinase-deficient TGF-B receptor II (TBRIIdeltaK). Contrary to our expectations, SSc fibroblasts were mainly unresponsive to this treatment with regard to collagen production. On the other hand, this treatment resulted in a significant downregulation of the ECM production in healthy skin fibroblasts. These results led to a revision of our hypothesis and prompted us to investigate alternative mechanisms that may be responsible for the SSc phenotype. To investigate TGF-B independent pathways, we have focused on CTGF (connective tissue growth factor) and IGFBP5 (IGF binding protein 5). The current research proposal is based on our novel observations indicating that CTGF induction of ECM in human fibroblasts is dependent on insulin signaling and that IGFBP5 also stimulates collagen production by fibroblasts. We propose the following Specific Aims to test the hypothesis that interactions between TGF-B, CTGF, and insulin/IGF pathways are involved in the regulation of the SSc phenotype. In Specific Aim 1 we will continue to examine the role of the components of the TGF-B signaling pathway in the manifestation of the SSc phenotype. In Specific Aim 2 we will determine the role of the CTGF-mediated pathway in ECM production by SSc fibroblasts. We will delineate the mechanism of CTGF stimulation of the COL1A2 promoter and characterize the components of the insulin/IGF signaling pathway that contribute to CTGF induction of collagen. In Specific Aim 3 we will determine the mechanism of IGFBP5 stimulation of collagen production by SSc and healthy fibroblasts. We will analyze expression patterns of IGFBPs in SSc and healthy fibroblasts and utilize purified IGFBP proteins and corresponding cDNAs to probe their role in collagen regulation by SSc and healthy fibroblasts. In Specific Aim 4 we will examine the in vivo expression of the TGF-B receptor subunits, CTGF, and IGF/IGFBPs in SSc skin.